Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae

Abstract

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane. We name this protein Atg36 as its absence blocks pexophagy, and its overexpression induces pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae.

Figure 1

Atg36 is required for pexophagy. Validation of Pex11–GFP as a marker for pexophagy in WT, atg1Δ, and atg36Δ cells by (A) microscopy and (B) western blot including pex14Δ cells. The fluorescence assay shows that like HcRed–PTS1, Pex11–GFP accumulates in the vacuole in starvation medium (A), which corresponds to the accumulation of GFP on a western blot (B). (B) Cells were grown 18 h in oleate medium and switched to glucose medium lacking nitrogen (SD-N) for the times indicated. GFP* indicates the relatively protease-resistant degradation product indicative of vacuolar breakdown. (C) Pexophagy assay by fluorescence of GFP–PTS1 expressed from the CTA1 promoter in WT, atg36Δ, atg1Δ, and pep4-3, prb1-1122 strains. Cells were grown as above for Pex11–GFP pexophagy assay and imaged after 22 h in SD-N medium. Multiple fluorescent images were acquired in Z-axis and flattened into a single image. Bright-field image is a single plane. (D) FM4-64 labelling of cells grown as in (C). The three central images of a Z-stack were flattened into a single plane. Bright-field image is a single plane. Bar, 5 μm. (E) Pexophagy as assayed by Pex11–GFP western blot of cells grown for up to 3 days in glucose, oleate or glycerol medium. Samples of WT cells (left panel) and atg36Δ cells (right panel) were taken from the cultures after 24, 48 and 72 h.

Figure 2

Atg36 is not required for the Cvt pathway, mitophagy or non-specific autophagy. (A) Cvt pathway activity was assessed by endogenous expression of Ape1–GFP in WT, atg36Δ and atg1Δ cells. Cells were grown for 18 h in glucose medium and vacuoles were stained with FM4-64. Accumulation of GFP in the vacuole indicates normal function of pathway. Bar, 5 μm. (B) Mitophagy of WT, atg36Δ and atg1Δ cells was assayed by western blot analysis of OM45–GFP after culturing cells for up to 3 days in glycerol medium. Samples were taken at 24 h intervals. (C) WT, atg36Δ and atg1Δ cells were assayed for non-specific autophagy by alkaline phosphatase assay. Cells were grown in YPD and shifted to SD-N medium for 4 h. Samples were collected and processed for Pho8Δ60 activity. The results represent the mean and s.d. of three experiments. WT 4 h starvation is set at 100%.

Figure 3

Atg36 localises to peroxisomes and induces their autophagic degradation. (A) N- (left) and C- (right) GFP-tagged ATG36 under control of the GAL1 promoter was induced in atg36Δ or atg1Δ cells. Early (2 h) and later (5 h) time points after induction are shown. Pexophagy is assessed using the peroxisomal marker HcRed–PTS1 expressed from the constitutive HIS3 promoter. (B) Pex11–GFP western blot showing pexophagy in WT cells grown 6 h in galactose medium (0) then switched to starvation medium for 1, 2 or 3 h as indicated. (W), WT (empty plasmid); (N), GAL1–mRFP–ATG36; (C), GAL1–ATG36–mRFP; (U), GAL1–ATG36. WT shows pexophagy by endogenous Atg36. (C) Pex11–GFP pexophagy of WT cells containing either empty plasmid (WT) or GAL1–mRFP–ATG36 grown 18 h in oleate medium (0) then switched to oleate medium containing 2% galactose and harvested at 3, 6 or 22 h as indicated.

Figure 4

Atg36 binds peroxisomes via Pex3. (A) An in vitro quick-coupled transcription/translation assay was performed using E. coli-produced GST–Pex3 (40–441) and GST bound to Glutathione Sepharose beads. After extensive washing of the bound protein, Atg36 synthesised in vitro in the presence of [³⁵S]-Methionine was added. Following further washing, protein was eluted with GSH elution buffer and subjected to SDS–PAGE. Bound fractions and the Atg36 input were analysed by Coomassie staining (bottom panel) and PhosphoImaging (top panel). A beads-only sample was used as a further control. *Represents bound GST–Pex3 (40–441) and **represents bound GST. (B) atg36Δpex3Δ cells were transformed with plasmids encoding GAL1–GFP–Atg36 and a GAL1–Tom70–Pex3–mRFP chimeric protein (left panels, top) or GAL1–GFP–Atg36 (right panel, top). Expression was induced for 3 h. WT cells were transformed with GAL1–Tom70–Pex3–mRFP and preCox4–GFP (Motley et al, 2008) to confirm the mitochondrial localisation of Tom70–Pex3–mRFP. Bar, 5 μm. (C) Split-GFP analysis in atg8Δ cells. Cells expressing GFP halves were grown for 4 h in galactose medium, and scored for presence of fluorescence (−, no signal; +, fluorescent signal). Peroxisomes in cells expressing GFP–C-Atg36 plus Pex3–GFP-N were identified by mating with pex19Δ cells expressing HcRed–PTS1. Multiple fluorescent images were acquired in Z-axis and flattened into a single image. Bright-field image is a single plane. Bar, 5 μm. (D) Coimmunoprecipitation of Pex3 with Atg36. IP was performed in background strains of C13 abyss or C13–Atg36–PtA using plasmids expressing Pex3–GFP or Pex13–GFP under control of their endogenous promoters. Cells grown for 24 h to post-log phase in glucose medium. IgG Sepharose beads were used to immobilise Atg36–PtA from spheroplast yeast lysates. SDS–PAGE gels were probed with anti-GFP and PAP. Yeast lysates represent 5% of the lysate added to the beads and analysed by immunoblotting. TL, total lysate; IP, immunoprecipitate.

Figure 5

The pex3-177 allele is affected in pexophagy. (A) Pex11–GFP pexophagy assayed by microscopy in pex3Δ cells and pex3Δatg1Δ cells transformed with plasmids containing either WT PEX3 or pex3-177, or with an empty plasmid ycplac111. Cells were grown in glucose, transferred to oleate medium for 18 h and switched to SD-N medium for 22 h. (B) Western blot analysis of same cells during starvation on SD-N medium. GFP* indicates the relative protease-resistant degradation product and reflects vacuolar breakdown. Time points for western blots indicated. (C) Pexophagy was assayed by Pex13–GFP breakdown in pex3Δ cells carrying plasmids as above. (D) Analysis of Atg36 localisation in pex3Δ cells expressing PEX3, pex3-177 and pex3-1. The mother cells of pex3-1 are devoid of peroxisomes (black arrowhead). (E) Coimmunoprecipitation of Pex3-177 with Atg36. IP was performed in the background strain of C13–Atg36–PtA using a plasmid expressing Pex3-177–GFP under control of its endogenous promoter. Cells grown for 24 h to post-log phase in glucose medium. IgG Sepharose beads were used to immobilise Atg36–PtA from spheroplast yeast lysates. SDS–PAGE gels were probed with anti-GFP and PAP. Yeast lysates represent 5% of the lysate added to the beads and analysed by immunoblotting. TL, total lysate; IP, immunoprecipitate. Comparison to Pex3–GFP IP (left panel).

Figure 6

Atg36 expression and cleavage correlates with Pex11–GFP degradation. (A) WT cells expressing genomic Atg36–PtA and Pex11–GFP on a plasmid were grown in glucose for 22 h, transferred to oleate medium, and then shifted to SD-N medium. Samples were taken at the times indicated and processed for western blot with peroxidase anti-peroxidase complex to detect Atg36–PtA (top panel) or anti-GFP to detect Pex11–GFP pexophagy (bottom panel). GFP* indicates the relative protease-resistant degradation product and reflects vacuolar breakdown. (B) Western blot analysis of genomic Atg36–GFP under oleate and starvation conditions for the indicated time points in the strains indicated. (C) Western blot analysis of genomic Atg36–GFP in WT, pex14Δ, atg1Δ and pex3Δ cells grown for up to 3 days in oleate medium. Samples were taken at 24 h intervals. (D) Pex3 level in total lysate was determined from WT cells grown in glucose medium for 22 h, transferred to oleate medium and then shifted to SD-N medium. Samples were taken at the times indicated and processed for western blot with anti-Pex3. A pex3Δ glucose 22 h sample was used to determine the specificity of the antibody. **Indicates non-specific band. (E) Western blot analysis of pex3Δ and atg36Δpex3Δ cells expressing Pex3–GFP from its own promoter after growth in glucose, oleate-containing medium and after shifting to SD-N medium for the times indicated.

Figure 7

Atg11 and Atg8 bind Atg36. (A) Coimmunoprecipitation of Atg11 and Atg8 with Atg36. IP was performed in either C13–Atg36–PtA atg1Δ or C13–Mvp1–PtA atg1Δ cells containing plasmids expressing either HA–Atg11 or HA–Atg8 under control of the TPI1 promoter. Cells were grown for 24 h to post-log phase in glucose medium followed by growth in oleate medium for a further 18 h. Cells were then grown under non-starvation or starvation conditions for a further 2 h. IgG Sepharose beads were used to immobilise Atg36–PtA or Mvp1–PtA from spheroplast yeast lysates. After extensive washing, bound material was eluted by Tev protease cleavage. SDS–PAGE gels (percentage indicated) were probed with anti-HA (monoclonal 12CA5) and PAP. TL, total lysate; IP, immunoprecipitate (Tev eluate). (B–D) GFP–Atg11 was expressed in strains as indicated and cells were grown to post-log phase in glucose medium. Inset shows magnification plus individual slices of stack from atg1Δ cells. Bar, 5 μm.

Figure 8

Atg36 drives mitophagy when directed to mitochondria. (A) pex3Δ, pex3Δatg1Δ and pex3Δatg32Δ cells were grown for 6 h in galactose medium to express GAL1–Tom70–Pex3–mRFP and GAL1–Atg36–GFP. Cells were shifted to SD-N medium and imaged after 12 h. Multiple fluorescent images were acquired in Z-axis and flattened into a single image. Bright-field image is a single plane. Bar, 5 μm. (B) OM45 was tagged with GFP in the genome of atg32Δpex3Δ and atg32Δpex3Δatg36Δ cells, which were transformed with an empty plasmid (−) or a plasmid encoding OM45–Pex3 (+). The cells were grown for up to 3 days in glycerol medium and samples were taken at 24 h intervals and processed for anti-GFP western blotting. GFP* indicates the relative protease-resistant degradation product and reflects vacuolar breakdown.